Use of Shigella invaplex to transport functional proteins and transcriptionally active nucleic acids across mammalian cell membranes in vitro and in vivo

ABSTRACT

The in vivo and in vitro use of Invaplex to transport materials, including functional proteins and biologically active nucleic acids, across eukaryotic cell membranes. The eukaryotic cells include a variety of cell types, e.g. insect, reptile, fish, mammal and tumor cells. The suitable materials for transport include biochemicals such as reporter molecules, antibiotics, biopharmaceuticals and carbohydrates including polysaccharides, lipopolysaccharides, polynucleotides, such as DNA and RNA, and glycoproteins and proteins including antigens, enzymes, antibodies, receptors and hormones. In addition, Invaplex enhances the immune response to DNA vaccines and also can function by itself as a vaccine against shigellosis.

This application is a continuation of application Ser. No. 10/994,463,filed Nov. 23, 2004, now U.S. Pat. No. 7,632,659, which is based onprovisional application Ser. No. 60/524,639 filed on Nov. 25, 2003. Thecontent of the provisional application are expressly incorporated hereinby reference.

FIELD OF THE INVENTION

The present invention provides for the in vivo and in vitro use ofInvaplex to transport materials including functional proteins andbiologically active nucleic acids across mammalian cell membranescompositions. The present invention also relates to adjuvants,immunogenic compositions and methods useful for polynucleotide-basedvaccination.

BACKGROUND OF THE INVENTION

Invaplex as a Vaccine

The pathogenesis of Shigella spp. is attributed to this organism'sability to invade, replicate intracellularly, and spread intercellularlywithin the colonic epithelium. Several highly conserved, virulenceplasmid-encoded proteins, called the invasion plasmid antigens (IpaA,IpaB, IpaC, and IpaD) (1), are essential participants in the invasionprocess. Upon contact or attachment to host cells, the Shigella invasinsare released (22) by a type III secretion apparatus (8) and induce aphagocytic event resulting in engulfment and internalization of thebacterium by the host cell (7). The active components include anIpaB:IpaC complex that integrates into the host cell membrane, forming achannel by which other Shigella proteins gain entry into the host cell(16).

Recently, we have isolated an invasin protein-LPS complex from intact,virulent Shigella cells (20). The invasin complex or Invaplex is thesubject of several issued or pending WRAIR patents (11-14). The invasincomplex (Invaplex) binds to surfaces of epithelial cells and quicklybecomes internalized, presumably by an endocytic process (Oaks andKaminski, unpublished data). The ability to bind to a eukaryotic hostcell surface and induce a phagocytic event indicates that the Invaplexmaintains an active, native virulence structure similar to that found onthe surface of invasive Shigella. In fact, many of the key, antigeniccomponents found in Invaplex 24 and Invaplex 50 (see below) are locatedon the Shigella surface. These antigens include IpaB, IpaC, IpaD and LPS(all of which are present in both Invaplex 24 and Invaplex 50) and alsothe newly described protein antigens 72 kDa, 84 kDa, and a 60 kDaprotein found exclusively in Invaplex 50 (Oaks & Turbyfill, unpub data).More recently a highly purified form of Invaplex (HP Invaplex) has beenisolated by size-exclusion chromatography (SEC). The HP Invaplex 24consists of IpaB, IpaC and LPS and has an estimated mass of about 1MDal. Experiments in mice have determined that the S. flexneriHP-Invaplex 24 or HP-Invaplex 50 offer enhanced immunogenicity andefficacy over the parent Invaplex indicating that the active componentsof Invaplex are, at a minimum, IpaB, IpaC and LPS.

The ability to isolate a putative native surface structure such asInvaplex, which exhibits activities and immunogenicity similar toinvasive shigellae, has significant implications in vaccine design anddevelopment. First, the putative native structure may enhance deliveryto the appropriate portal of entry (M-cells), similar to that targetedby live-attenuated vaccine strains. In the case of Invaplex, this hasallowed the use of relatively small doses for intranasal immunizationdue to its delivery efficiency. Similar to live, attenuated vaccines,immunization with the isolated subcellular, native Invaplex structurethat contains all known Shigella antigens results in an immune responseequivalent to that produced during natural infection, includingrecognition of epitopes found only in native structures. The immunitystimulated by Invaplex is highly protective in mice and guinea pigs (12,20).

Invaplex as an Adjuvant

The delivery of antigens in a manner, which safely stimulates aprotective mucosal immune response, is critical to the successfuldevelopment of enteric vaccines. As an alternative to live attenuatedvaccines, which are often difficult to construct, standardize anddeliver without the risk of side effects, subunit vaccines offer thepromise of chemically defined, well-standardized products.Unfortunately, the rapid increase in potential subunit vaccines arisingfrom recombinant, synthetic, or subunit purifications, including bothprotein and DNA vaccines, has outpaced the ability to deliver thesenovel vaccines safely and effectively. A major obstacle has been thefailure to develop an adjuvant, which effectively stimulates the mucosalimmune system in a safe, nontoxic manner. One highly effective mucosaladjuvant is cholera toxin (CT), another is E. coli heat-labileenterotoxin (LT); unfortunately, both are extremely toxic molecules thatrequire substantial detoxification by genetic manipulations. Beforegenetically modified forms of CT or LT become available as adjuvants forhuman use, they will require extensive safety testing.

A unique property of the Invaplex is that it enhances the immuneresponse to substances that are not very immunogenic (11). Theadjuvanticity of Invaplex has been demonstrated with several proteinsincluding ovalbumin, the recombinant Sta56 (56K) protein of Orientiatsustsugamushi, the FlaA protein of Campylobacter jejuni, colonizationfactors of enterotoxigenic E. coli and the PA (protective antigen) ofBacillus anthracis (Oaks and Kaminski, unpublished data). Theimmunogenicity and adjuvanticity of the Invaplex is likely due to itsability to target and induce uptake by immune cells, possibly M cellequivalents in the mucosa. Stimulation of a mucosal immune responseoften requires uptake of the antigen or pathogen by M cells in the gutor comparable cells in other mucosal tissue. The M cells lie over anarea of cells called the mucosa associated lymphoid tissue (Peyer'spatches in the gut). Upon uptake of antigen, the M cell is capable oftranslocating the antigen to the lymphoid tissue consisting oflymphocytes, macrophages, and dendritic cells. These cells serve topresent the antigen to antigen-specific lymphocytes resulting instimulation, expansion, and expression of specific immune effectors. Inthe mucosa, this process leads to the development of IgA-producing Bcells.

The adjuvanticity of Invaplex is likely mediated by IpaB and IpaC, whichare crucial virulence proteins involved in the invasiveness ofshigellae. Very little is known about the effect Invaplex has on hostcells.

DNA Transfection and DNA Vaccines.

The process of delivering transcriptionally active DNA into eukaryoticcells is called transfection. The result of transfection is heterologousgene expression in vitro or in vivo. Transfection is often used as ameans to study the function of specific proteins expressed in thetransfected cell. Although physical methods such as microinjection andelectroporation can be used to shuttle DNA into eukaryotic cells,methods more amenable to in vivo work have been developed. Transfectionof cells in vivo has extended the in vitro functional analysis into liveanimals and has also allowed the immunogenicity of the expressed proteinto be evaluated if the levels of antigen expression in vivo are highenough. One advantage of DNA vaccines is that the DNA can be producedeasily and is relatively inexpensive (2). Optimal expression of thegenes of interest often requires genetic customization of the clonedgene. In DNA vaccines, in particular those delivering bacterial antigengenes, a cloned eukaryotic promoter, such as the cytomegalovirus (CMV)promoter, is used to drive expression of the antigen gene. Otherconsiderations of the antigen genes are codon usage (certain bacterialgenes may be suboptimal for eukaryotic expression) and the stability ofthe DNA construct in particular during delivery at mucosal sites. ManyDNA vaccines have been delivered intramuscularly or intradermally withthe gene gun (21). Mucosal delivery of DNA vaccines is difficult due tothe likely degradation of the DNA upon exposure to enzymes and harshconditions in the mucosa. Mucosal DNA delivery systems include liposomes(4, 6), microparticles (3) and live bacterial vectors (5). None of thesemucosal delivery systems use a native acellular bacterial product, suchas Invaplex, to deliver the DNA.

Protein Delivery Systems

In most cases a successful DNA delivery system (transfection reagent) issomewhat universal in that it will work, with most DNA molecules due tothe relatively similar biochemical (negatively charged nucleic acid)structure of DNA. Proteins, on the other hand, have a much more variedbiochemical structure, in that their net charge, conformation,hydrophilicity, and size are highly variable. This creates a differentproblem for transporting functional proteins into host cells. Strategiesused to transport proteins into cells include the use of cationic lipids(23) or specialized peptides consisting of protein transduction domains(17) or membrane transport signals (15). The specialized peptidescontain a high proportion of positively charged arginine and lysineresidues which are thought to interact with the cell membrane therebyinitiating the uptake of the desired protein. Other mechanisms forprotein delivery include microinjection and electroporation. Ideally anoptimal protein transport reagent would be useful for a variety ofproteins and target cells and would not exert significant toxicity onthe target cell. A universal protein transport system using a nativeacellular bacterial product, like Invaplex, has not been described.

SUMMARY OF THE INVENTION

The present invention provides for the in vivo and in vitro use ofInvaplex to transport materials, including functional proteins andbiologically active nucleic acids, across eukaryotic cell membranes. TheInvaplex can be in a composition form which would include thebiologically active material, e.g. compound, of interest and theInvaplex in an amount sufficient to cause a eukaryotic cell to take upthe compound. This composition can be placed in a kit wherein thecomposition is placed in a container. If desired, the Invaplex and thematerial of interest can be placed in separate containers in the kit.The kit can contain the materials in dosage amounts for a singleapplication, if desired. The kit can contain additional reagents andinstructions for use.

The invention also includes a process wherein the compound of interestand Invaplex are placed in close proximity to the eukaryotic cellmembrane. The eukaryotic cell is contacted with the material and asufficient amount of Invaplex to cause the cell to take up the material.

Invaplex is non-toxic to eukaryotic cells and induces endocytosis, whichstimulates the uptake of nearby materials. Invaplex adheres to mammaliancell membranes and is internalized by mammalian cells. Invaplex does notcause cytopathic effects in vitro at concentration ranges of to 60 to500 gg/ml. The eukaryotic cells can include a variety of cell types andsources, e.g. insect, reptile, fish, mammal and tumor cells.

The Invaplex complex is described in U.S. Pat. Nos. 6,277,379 and6,245,892, the contents of which patents are expressly incorporatedherein by reference.

In addition, Invaplex enhances the immune response to DNA vaccines andcan function as a vaccine against shigellosis by itself.

Transported materials include biochemicals such as vectors (e.g.,plasmids), reporter molecules, markers, antibiotics, antibodies,antigens, biopharmaceuticals, enzymes, receptors and hormones,carbohydrates including polysaccharides, lipopolysaccharides,polynucleotides, such as DNA and RNA, and glycoproteins, proteins, andpeptides.

Intranasal delivery of DNA combined with Invaplex is a simple,noninvasive means for immunization that does not require swallowing orinjection. Further the Invaplex delivery system does not require geneticmanipulation, as would be required for live attenuated strains carryingvaccine DNA. The system is easily adapted to many different antigensystems. The formulation is a matter of mixing of the target DNA withInvaplex prior to immunization.

The invention has a variety of uses including but not limited totherapeutic uses including immunological based therapies, vaccines, genetherapy; research tool uses including genetic manipulation includingchanges in phenotypes and genotypes cell sorting; and manufacture ofbiologics including biopharmaceuticals and the like clinical anddiagnostic.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-D show S. flexneri 2a Invaplex-Induced Cytotoxicity Assay. FIG.1A shows untreated cells stained with Evan's Blue. FIG. 1B shows BHK-21cells incubated overnight with S. flexneri 2a Invaplex-24 and stainedwith Evan's Blue. FIG. 1C shows BHK-21 cells incubated overnight with S.flexneri 2a Invaplex-50 and stained with Evan's Blue. FIG. 1D showsBKH-21 cells incubated overnight with GenePorter transfection reagentand stained with Evan's Blue.

FIGS. 2A and B show, respectively, the adherence of mammalian cellmembranes with S. flexneri 2a Invaplex 24 (FIG. 2A) or with Invaplex 50(FIG. 2B). FIG. 2C shows the internalization of Invaplex within thecell.

FIG. 3(A-F) show S. sonnei Invaplex 24 and 50 adherence to BHK-21fibroblast cells.

FIG. 4(A, B) show Invaplex S. sonnei 24 interacting with BHK-21fibroblast cell.

FIG. 5 shows Invaplex S. sonnei 50 interacting with BHK-21 fibroblastcell.

FIG. 6(A-F) show S. flexneri Invaplex-24 located within early and lateendosomes.

FIG. 7(A, B) show S. flexneri 2a Invaplex-mediated GFP plasmidtransfection.

FIG. 8(A-C) show S. flexneri 2a Invaplex-Mediated beta-Galactosidaseplasmid transfection.

FIG. 9(A-F) show Invaplex-Mediated Transport of Green FluorescentProtein across mammalian cell membrane.

FIG. 10(A-C) show Invaplex-Mediated Transport of beta-Galactosidaseprotein across mammalian cell membrane.

FIG. 11(A-F) show Invaplex-mediated transfection of plasmid DNA encoding56k protein and transport of 56k protein intracellularly.

FIG. 12 shows Anti-56k protein serum IgG responses detected afterDNA-Prime-Protein-Boost Strategy with Invaplex formulated vaccines.

FIG. 13 shows Lymphoproliferative responses in splenocytes stimulatedwith r56k in vitro.

FIG. 14(A-C) show proliferative responses in murine splenocytesstimulated in vitro with Con A, S. flexneri 2a IVP-50 or Sta56 protein.Antigen-specific proliferation was measured after five days of cultureusing a non-radioactive cell proliferation assay. Data is expressed asthe mean stimulation index of each vaccine group. Error bars represent±1 SEM. A reference line denotes a stimulation index of 2. Vaccinegroups are indicated on the horizontal axis.

FIG. 15(A-B) show Anti-Sta56 and anti-S. flexneri 2a IVP-50 serum IgGresponses in mice intranasally immunized with plasmid DNA encoding thesta56 gene from O. tsutsugamushi administered alone, or in combinationwith S. flexneri 2a IVP-50 and boosted with purified Sta56 proteincombined with S. flexneri 2a IVP-50.

DETAIL DESCRIPTION OF THE INVENTION

In vitro observations suggest that the Invaplex interacts with host-cellmembranes eventually being translocated into the cytoplasm. This eventis indicative of an induced endocytic event similar to the activityexpressed by virulent shigellae. A hypothesis was that if a heterologousmolecule (DNA or protein) is present at the time of Invaplex-inducedendocytosis, the heterologous molecule would be taken up,non-specifically, by the host cell. Without the Invaplex stimulationsuch uptake would not occur. This has been demonstrated forInvaplex-induced uptake of naked DNA (expressing GFP, B-galactosidase orthe Sta56 protein) or purified proteins (pure GFP, pure B-galactosidaseor Sta56) by BHK cells, indicating that the Invaplex may be useful fordelivering prophylactic or therapeutic molecules across eukaryotic cellmembranes, thereby providing a safe and effective method fortransfecting cells. The data below documents the interaction of Invaplexwith host-cells, to Invaplex-induced uptake of DNA and proteins, andfinally DNA immunization of mice using Invaplex to deliver the DNA.

EXAMPLES

Isolation and Characterization of Invaplex

Purification of Shigella Invasin Complex (Invaplex).

The Shigella Invaplex was isolated from water extracted proteins ofvirulent Shigella including Shigella flexneri 2a (2457T) and S. sonnei(Mosely). A modification of the original water extraction procedure (10)was used to prepare the material from which the Shigella invasin complexis isolated (20). Water extract batches positive for IpaB and IpaC wereapplied to an anion exchange FPLC (HiTrapQ, Pharmacia) column. Two peaks(measured at 280 ηm) containing the invasins and LPS were collected. Thefirst peak (Invaplex 24) is eluted with 0.24M NaCl in 20 mM Tris HCl, pH9.0 and the second peak (Invaplex 50) is eluted with 0.50 M NaCl in 20mM Tris-HCl, pH 9.0. Each FPLC fraction was analyzed by immuno-spot blotusing monoclonal antibodies (9) for the presence of IpaC and IpaB.Fractions containing the greatest amount of IpaB and IpaC in theInvaplex 24 peak were pooled as were peak Ipa protein fractions in theInvaplex 50 peak. The final pools were aliquoted and stored at −80° C.

S. flexneri 2a Invaplex-Induced Cytotoxicity Assay

BHK-21 cells (ATCC CCL-10), 5×10⁴ cells per well in Minimum EssentialMedia (MEM) (Gibco, Rockville, Md.) supplemented with 1% L-glutamine(L-glut) (Gibco, Rockville, Md.) and 7% fetal calf serum (FCS) (Sigma,St. Louis, Mo.), were cultured overnight at 37° C., 5% CO₂ in a 96-welltissue culture plate (Costar, Acton, Mass.). S. flexneri 2a Invaplex 24and Invaplex 50 (both from lot GNGO) were diluted with sterile,deionized water to isotonic levels (0.15M NaCl) and concentrated viaUltrafree-MC 30,000 MWCO centrifuge filters (Millipore, Bedford, Mass.).Final protein concentration was determined by bicinchoninic acid proteinassay originally described by Smith et al (Pierce, Rockford, Ill.) (18).S flexneri 2a Invaplex 24 and 50 were then titrated, using MEMsupplemented with 1% L-glut without FCS as the diluent in duplicatewells and incubated with the BHK-21 cells for 90 minutes at 37° C.Concentrations of Invaplex ranged from 500 μg/mL to 60 μg/mL.

A non-radioactive cytotoxicity assay (CytoTox 96® from Promega, Madison,Wis.) was used to determine release of lactate dehydrogenase (LDH), astable cytosolic enzyme that is released from the cell's cytoplasm uponcell lysis or cell membrane damage. 10 μL of lysis buffer (9% v/v TritonX-100) was added at 90 minutes to non-Invaplex additional 30 minutes.The plate was centrifuged at 250×g for 5 minutes and 50 μL ofsupernatant from each well was transferred to a separate plate. 50 μL ofLDH substrate, containing the tetrazolium salt INT (iodonitrotetrazoliumchloride), was added to each well, covered from light and incubated at37° C. for 30 minutes at which time 50 μL of stop solution (IM aceticacid) was added to each well and the absorbance was measured at 492 ηm.All samples were run in duplicate.

The percentage of cells exhibiting cytotoxic effects was calculated asthe experimental LDH release (O.D. 490 ηm) divided by the maximum LDHrelease determined by the cells treated with lysis buffer (O.D. 490 ηm)multiplied by 100.

Adherence of S. flexneri 2a Invaplex 24 and 50 with Mammalian CellMembranes

BHK-21 cells, diluted to 5×10³ cells per well, were cultured in MEMsupplemented with L-glut and 7% FCS overnight at 37° C., 5% CO₂, in8-well, sterile, Lab Tek II Chamber Glass Slides with cover (Nalge NuncInternational, Naperville, Ill.). Cells were washed 3 times withphosphate buffered saline (PBS) and incubated with either isotonic S.flexneri 2a Invaplex 24 (66.5 μg/well) or Invaplex 50 (27.7 μg/well)(both from lot GNGO) at 37° C., 5% CO₂. After 5, 10 and 30 minutes ofincubation, two of the eight wells were selected and were washed 2 timeswith PBS and the cells fixed with 100% methanol. Negative control wellscontaining BHK-21 cells incubated with only cell culture media, werefixed at 30 minutes. All wells were then incubated at room temperaturefor 60 minutes with rabbit anti-Shigella flexneri 2a (strain 2457T)serum diluted 1:100 in PBS. Cells were then washed 3 times with PBS andincubated for 60 minutes at room temperature with Protein A-FITC diluted1:1000 in PBS. Cells were washed 3× with PBS, once with deionized water,allowed to air dry, and viewed under a Nikon Optiphot 2 microscope usingthe EX470-490 excitation filter at 100× magnification.

Adherence of S. sonnei Invaplex 24 and 50 with Mammalian Cell Membranes

BHK-21 cells, diluted to 5×10³ cells per well, were cultured in MEMsupplemented with L-glut and 7% FCS overnight at 37° C., 5% CO₂ in 8well chamber slides. Cells were washed 3 times with PBS and incubatedwith either isotonic S. sonnei (Mosley) Invaplex 24 or Invaplex 50 (50μg/well lot FRFT and lot HKHL, respectively) at 37° C. At 5, 10 and 30minutes of incubation, two of the eight wells were selected and thewells were washed 2× with PBS and cells fixed with 100% methanol.Negative control wells containing only BHK-21 cells incubated with mediawere fixed at 30 minutes. All wells were then incubated at roomtemperature for 60 minutes with mouse polyclonal anti-Shigella sonneiserum diluted 1:100 in PBS. Cells were then washed 3× with PBS andincubated for 60 minutes at room temperature with goat-anti-mouse-TRITC(KPL, Gaithersburg, Md.) diluted 1:100 in PBS. Cells were washed 3× withPBS, 1× with deionized water, allowed to air dry and viewed under aNikon Optiphot 2 microscope using the EX470-490 excitation filter at 20×magnification.

Transfection of Mammalian Cells with Plasmid DNA Encoding GreenFluorescent Protein (GFP) Using Shigella Invaplex as the TransfectionMediation Reagent.

BHK-21 cells, 5×10⁴ cells per well, were cultured overnight at 37° C.,5% CO₂ in 8 well chamber slides. 0.5 μg of a plasmid (pEGFP-N1) (GeneTherapy Systems, San Diego Calif.) encoding GFP with a cytomegalovirus(CMV) promoter was incubated for 30 minutes at room temperature witheither S. flexneri 2a Invaplex 24 or Invaplex 50 (both from lot GNGO)diluted to 10 μg/200 μL or 20 μg/200 μL in MEM and L-glut without FCS ina 1.5 ml eppendorf tube. As a positive control, the GFP plasmid was alsoincubated with GenePorter (Gene Therapy Systems, San Diego, Calif.)reagent as per manufacturer's directions. Negative controls includedcell media only and plasmid pEGFP-N1 but no transfection reagent. After30 minutes incubation, all wells were washed 3 times with MEMsupplemented with L-glut. Cells were then treated with 200 μL of amixture containing either the plasmid pEGFP-N1 and Invaplex 24 orInvaplex 50 (test samples); plasmid and GenePorter mixtures (positivecontrol); plasmid pEGFP-N1 without a transfection reagent (negativecontrol I); or with cell culture media alone in the appropriate slidechamber well. Cells were incubated for 3 hours, after which 100 μL ofmedia containing 20% FCS was added to each well and cells incubatedovernight at 37° C. Cells were then washed 3 times with PBS, fixed withmethanol, and viewed under a Nikon Optiphot 2 microscope using EX470-490excitation filter at 30× magnification.

The percent GFP positive cells was determined by first randomly choosinga field and counting the cells present using bright field microscopy.Fluorescent cells in the same field were then counted. The percentagecells positive for GFP expression was calculated by dividing the numberof green fluorescent cells by the total number of cells in a particularfield and multiplying by 100.

Transfection of Mammalian Cells with Plasmid DNA EncodingBeta-Galactosidase Using Shigella Invaplex as the Transfection MediationReagent.

BHK-21 cells, 5×10⁴ cells per well, were cultured overnight at 37° C.,5% CO₂ in 8-well chamber slides. Plasmid DNA (0.5 μg) encodingbeta-galactosidase (β-gal) linked to a CMV promoter (gWiz(β-galactosidase plasmid, Genetic Therapy Systems, San Diego Calif.) wasincubated for 30 minutes at room temperature with either S. flexneri 2aInvaplex 24 or S flexneri 2a Invaplex 50 (lot GNGO) diluted to 10 μg/200μL or 20 μg/200 μL in serum-free media. As a positive control the β-galplasmid was also incubated with GenePorter (Gene Therapy Systems, SanDiego Calif.) reagent as per manufacturer's directions. Negativecontrols included BHK cells with culture media only (no plasmid) and BHKcells with plasmid alone without a transfection reagent. After 30minutes incubation, cells were washed 3× with serum-free media and 200μL of the culture media, plasmid alone, plasmid-Invaplex,plasmid-GenePorter mixtures were added to the appropriate chamber. Cellswere incubated for 3 hours, after which time, 100 μL of media containing20% fetal calf serum (FCS) was added to each well and cells incubatedovernight at 37° C. Cells were then washed 3× with PBS, fixed withmethanol, and viewed by bright field microscopy.

The percentage of β-gal positive cells was determined by counting all ofthe cells present in a randomly chosen field using bright fieldmicroscopy. Blue cells in the same field were then counted. The percentβ-gal positive was calculated by dividing the number of blue cells bythe total number of cells and multiplying by 100.

Invaplex-Mediated Transport of Purified Recombinant GFP Protein AcrossMammalian Cell Membranes

BHK-21 cells, 5×10⁴ cells per well, were cultured overnight at 37° C.,5% CO² in 8-well sterile glass Lab Tek II Chamber Slides with cover(Nalge Nunc International, Naperville, Ill.). Cells were washed 1× withserum-free media and incubated with GFP protein at 1 μg/well and eitherisotonic S. flexneri 2a Invaplex 24 (66, 33, or 15 μg/well) or isotonicS. flexneri 2a Invaplex 50 (28, 14, or 7 μg/well) (both from lot GNGO)at 37° C., 5% CO² overnight. Cell were washed 3× with PBS and fixed with100% methanol, allowed to air dry and viewed under a Nikon Optiphot 2microscope and EX470-490 excitation filter at 30× magnification.

The total number of cells were counted in a randomly chosen field ofview in each well using bright-field microscopy and the number cells inthe same field exhibiting green fluorescence was determined with afluorescent microscopy. The percentage of GFP positive cells wasdetermined by dividing the number of fluorescent cells by the totalnumber of cells and multiplying by 100.

Invaplex-Mediated Transport of Beta-Galactosidase Protein AcrossMammalian Cell Membranes

BHK-21 cells, diluted to 5×10⁴, were cultured overnight at 37° C., 5%CO² in 8 well chamber slides. Cells were washed 1× with serum-free mediaand incubated with β-gal protein at 10 units/well (Sigma, St. Louis,Mo.) and either S. flexneri 2a Invaplex 24 (66, 33, or 15 μg/well) or S.flexneri 2a Invaplex 50 (28, 14, or 7 μg/well), both isotonic (lot GNGO)at 37° C., 5% CO² overnight. Media was aspirated and cells washed 1×with PBS. 500 μL of fixing buffer from X-gal staining kit (GeneticTherapy Systems, San Diego, Calif.) was added to each well for 10minutes at room temperature. Fixative was aspirated and cells washed 3×with PBS. 500 μL of freshly made X-gal staining buffer was added to eachwell and incubated for 90 minutes at 37° C. Cells were washed 3× withPBS (500 μL of PBS per well).

The total number of cells in a randomly chosen field of view werecounted in each well using a light microscope and the number of thosecells that were blue (β-gal positive) was determined. The percentage ofβ-gal positive cells was calculated by dividing the number of blue cellsby the total number of cells and multiplying by 100.

Inhibition of Invaplex Adherence with Shigella-Specific Antibodies

BHK-21 cells, diluted to 5×10⁴, were cultured overnight at 37° C., 5%CO² in 8 well chamber slides. Invaplex 24 (Lot GNGO, isotonic) wasincubated at room temperature for 60 minutes with dilutions ofantibodies specific for Shigella epitopes. Cells were washed 1× withserum-free media and incubated with each Invaplex/antibody combinationat room temperature for 30 minutes. Cells were then washed 2× with PBSand fixed with 100% methanol. The fixed BHK cells were then incubated atroom temperature for 60 minutes with rabbit anti-S. flexneri 2a (strain2457T) serum (diluted 1:100 in PBS), washed 3× with PBS and finallyincubated with Protein A-FITC diluted 1:1000 in PBS for 60 min. at roomtemperature. Cells were washed 3× with PBS, 1× with deionized water,allowed to air dry and viewed under a Nikon Optiphot 2 microscope andEX470-490 excitation filter at 20× magnification.

Inhibition of Shigella Invaplex Transfection Potential withShigella-Specific Antibodies

BHK-21 cells, diluted to 5×10⁴, were cultured overnight at 37° C. in 8well chamber slides. Plasmid DNA encoding GFP (Genetic Therapy Systems,San Diego, Calif.) was diluted in MEM supplemented with 1% L-glut to afinal concentration of 2 μg/100 μL. S. flexneri Invaplex 50 and S.flexneri Invaplex 24 was diluted to 25 μg/100 μL in MEM supplementedwith 1% L-glut. The diluted isotonic Shigella Invaplex was then mixedwith equal volumes of the diluted plasmid DNA. Antibodies specific forShigella IpaC, IpaB were added to the Invaplex-plasmid DNA mixtures at afinal dilution of 1:100 and incubated at room temperature for 30minutes. Media was aspirated from the BHK-21 culture slides and 200 μLof the Invaplex-plasmid DNA-antibody mixtures were added to the cells.Controls for the experiment included cells treated with plasmid DNA withand without S. flexneri Invaplex 24 or S. flexneri Invaplex 50 and cellstreated with MEM supplemented with L-glut. Cells were incubated with theadmixtures for 4 hours, 200 μL of MEM supplemented with 1% L-glut and20% FCS was added to each well, and the cells were incubated for anadditional 18 hours at 37° C. Cells were washed 3× with PBS and fixedwith 100% methanol, allowed to air dry and viewed under a Nikon Optiphot2 microscope and EX470-490 excitation filter at 30× magnification.

The total number of cells were counted in a randomly chosen field ofview in each well using bright-field microscopy and the number cells inthe same field exhibiting green fluorescence was determined with afluorescent microscopy. The percentage of GFP positive cells wasdetermined by dividing the number of fluorescent cells by the totalnumber of cells and multiplying by 100.

Invaplex-Mediated Transport of Orientia tsutsugamushi 56k Protein andPlasmid DNA Encoding 56k Protein Across Mammalian Cell Membrane In Vitro

BHK-21 cells, diluted to 5×10⁴ cells per well, were incubated overnightin four-well glass chamber slides. Isotonic S. flex 2a Invaplex-50 andInvaplex-24 was diluted to 200 ug/mL in serum-free MEM supplemented with1% L-glut. Plasmid DNA encoding the 56k protein was diluted to 500 ug/mLand purified 56k protein was diluted to 200 ug/ml and incubated withdiluted Invaplex for 30 minutes at room temperature. Cells were washedtwo times with PBS and 300 uL of Invaplex-plasmid DNA or Invaplex-56kprotein were added to wells 3 and 4 of the chamber slide. Equal amountsof plasmid DNA or purified 56k protein were added to well 2 while well 1received only 300 uL of MEM supplemented with 1% L-glut. Slidescontaining Invaplex-protein preparations were incubated for 30 minutesat 37° C., washed 3 times with PBS, and fixed with 100% methanol. Aftera three hour incubation period at 37° C., 500 uL of MEM supplementedwith 20% FCS and 1% L-glut was added to slides containingInvaplex-plasmid DNA preparations. These slides were then incubatedovernight at 37° C., washed 3× with PBS, and fixed with 100% methanol.All chamber slide wells were then incubated at room temperature with 300uL of mouse anti-56K ascites fluid (K13F88A; 11-5-87) diluted 1:100 inPBS, washed three times with PBS and subsequently incubated with goatanti-mouse IgG labeled with TRITC diluted 1:1000 in PBS. After threewashes with PBS and one wash with deionized water, slides were air-driedand viewed under an Nikon Optiphot-2 microscope using an EX546/10excitation filter at 100× magnification. Successful transport of 56Kprotein and transfection plasmid DNA encoding the 56K protein wasdetermined by the presence of intracellular red fluorescence.

Invaplex-Mediated Delivery of Naked Plasmid DNA Encoding the Orientiatsutsugamushi 56K Protein for Intranasal Immunization of Mice.

Mice were immunized intranasally on weeks 0, 2, and 4 with plasmid DNA(100 or 2 ug) encoding the Rickettsial 56k protein formulated with orwithout 10 of S. sonnei Invaplex 50.¹ Two of the five animals per groupwere immunized on week 8 with r56k protein (5 ug) formulated with S.sonnei Invaplex 50 (5 ug). Plasmid DNA was provided by Dr. Wei-Mei Chingof NMRC. ¹ Groups of mice were intranasally immunized on day 0, 14 and28 with plasmid DNA encoding the sta56 gene from O. tsutsugamushi linkedto a CMV promoter (pVR1012_(—)56K) alone or pVR1012_(—)56 K) (25 or 100μg) combined with S. flexneri 2a IVP-50 (15 μg). Other groups of micewere immunized with saline, S. flexneri 2a IVP-50 (15 μg), or emptyplasmid DNA vector (pVR 1012) combined with S. flexneri IVP-50 (15 μg).All groups were then intranasally immunized with recombinant Sta56protein (15 μg) co-delivered with S. flexneri 2a IVP-50 (10 μg) on day56. Blood was collected from all animals on days 0, 28, 35, 42, 56, 63,and 70. The Sta-specific (FIG. 15A) and S. flexneri IVP-50-specific(FIG. 15B) serum IgG responses were determined by ELISA. OD₄₀₅ valuesrepresent the mean OD₄₀₅ at a 1:180 (FIG. 15A) or 1:2880 (FIG. 15B)dilution of sample after a 60-minute incubation with substrate for eachgroup of mice (n=5/grp).

Blood was collected at weeks 0, 4, 5, 9, 10, and 11 from each animal andcells from the spleen and cervical lymph nodes were collected from threeof the five animals per group at week 6 upon sacrifice. Blood sampleswere assayed for anti-56k IgG responses using ELISA. Antigen-specificproliferative responses in cells from spleens and cervical lymph nodeswere assessed using a colorimetric cell proliferation assay.

Detection of r56K Protein-Specific Serum Antibodies by ELISA

Detection of systemic antigen-specific antibodies was assessed by enzymelinked immunosorbant assay (ELISA). Recombinant 56k protein was dilutedto 3 ug/mL in carbonate coating buffer (0.2 M carbonate, pH 9.8) andadded to polysytrene 96-well antigen plates (0.3 ug/well) (DynexTechnologies, Inc. Chantilly, Va.). After overnight incubation at 4° C.,plates were blocked for 30 minutes with casein (2% casein in Tris-salinebuffer, pH 7.5). Serum samples were diluted in 2% casein, added to theantigen-coated plates, and incubated at room temperature for 2 hours.After four washes in phosphate-buffered saline (10.75 mM sodiumphosphate, 145 mM NaCl, pH 7.4) with 0.05% Tween 20, plates wereincubated with anti-mouse IgG conjugated with alkaline phosphatase(Kirkegaard & Perry, Gaithersburg, Md.). After incubation, plates werewashed four times as above and the substrate, para-nitrophenyl phosphate(1 mg/ml in 10% diethanolamine buffer, pH 9.8 containing M_(g)Cl₂ (0.1mg/ml) and 0.02% sodium azide), was added to each well. Optical densitywas measured at 405 nm on a Molecular Devices (Menlo Park, Calif.) ELISAplate reader.

Antigen-Specific Lymphoproliferation Assays

Splenocytes were evaluated for antigen-specific lymphoproliferation byculturing lymphoid cells in complete media composed of RPMI 1640supplemented with L-glutamine 4 mM, penicillin (100 U/ml), streptomycin(100 mg/ml), β-ME (5×10⁻⁵ M) and 10% heat-inactivated FCS. Proliferativeresponses to antigens and mitogens were measured by incubating 1×10₅cells per well in 96-well U-bottom with either 5 or 1 μg of r56k, or S.flexneri 2a Invaplex 50. A subset of the cells were stimulated withconcanavalin A as a positive control. Negative controls included immunecells incubated with complete medium alone and cells from naive micestimulated with antigen. Assays were performed in triplicate and plateswere incubated at 37° C. in 5% CO₂.

Lymphoproliferation was assessed after 3-5 days of culture using anon-radioactive cell proliferation assay (CellTiter 96® Aqueous Assay,Promega) as per the manufacturer's directions. Briefly, plates werecentrifuged at 250×g for 5 minutes and 100 uL of cell supernatant wastransferred to a new flat bottom, 96-well microtiter plates and storedat −70° C. until assayed for cytokine concentrations. 20 μL, of theMTS-PMS reagent was added to the remaining 100 uL of supernatant and theplates were incubated at 37° C. for 1-4 hours. Absorbance at 492 nm wasmeasured after adding 25 μL of 10% SDS to stop the reaction.

Stimulation indices were calculated by dividing the mean optical densityrecorded in wells with antigen-stimulated cells by mean optical densityrecorded in wells with medium-only stimulated cells (19). Thestimulation index (SI) of cells from mice immunized with adjuvant andantigen were compared to the SI of cells from non-immunized mice, andmice immunized with antigen alone or adjuvant alone.

Results Section

Invaplex-Induced Cytotoxicity Assay

Neither Invaplex 24 nor Invaplex 50 displayed measurable cytotoxiceffects as determined by LDH release in the concentration range of 6 to50 μg/100 μL. Background levels of cytotoxicity as determined with mediaonly controls were also observed with Invaplex treated cells. Incontrast cells treated with GenePorter exhibited a much higher level ofcytotoxicity as determined by LDH release. (See Table 1.)

This data is consistent with microscopy observations made during thisand other assays in which no detectable morphological changes appear inBHK-21 cells incubated with Invaplex for extended periods of timeranging from 4 to 24 hours (FIG. 1A-D). In contrast, cells incubatedwith GenePorter transfection reagent underwent significant morphologicalchanges often resulting in lower proportion of viable cells.

TABLE 1 Percent Cytotoxicity of Invaplex-Treated Cells Amount % Sample(μ) Cytotoxicity Lysis buffer (9% Triton-X) 10 μL 100.00 Media Only N/A5.99 GenePorter 1:4 dilution 31.3 GenePorter 1:8 dilution 16.0 S.flexneri 2a Invaplex 24 50 4.27 S. flexneri 2a Invaplex 24 25 6.16 S.flexneri 2a Invaplex 24 12.5 6.16 S. flexneri 2a Invaplex 24 6 7.27 S.flexneri 2a Invaplex 50 50 4.27 S. flexneri 2a Invaplex 50 25 6.59 S.flexneri 2a Invaplex 50 12.5 6.78 S. flexneri 2a Invaplex 50 6 5.85Adherence of S. flexneri 2a Shigella Invaplex with Mammalian CellMembranes

BKH-21 cells were incubated with Invaplex 24 (FIG. 2 a) or with Invaplex50 (FIG. 2 b) for 5 minutes at 37° C. or with Invaplex 24 for 30 minutes(FIG. 2 c). Cells were then incubated with rabbit anti-S flexneri serum,washed, and subsequently incubated with Protein A conjugates with FITC.Invaplex location determined by recording green fluorescence with NikonOptiphot 2 and EX470-490 excitation filter at 60× magnification.

No observable fluorescence was noted in media only and Invaplex-freecontrols indicating that the fluorescence observed in each well wasInvaplex-associated. The level of fluorescence was similar for Invaplex24 and Invaplex 50 preparations despite the fact that approximately 50%less Invaplex 50 was used as compared to the Invaplex 24 in theseexperiments.

Invaplex-Specific Staining Pattern on the Host Cell Surface

Small fluorescent patches were noted after a 5-minute incubation asbright, semi-round decorations or patches localized at several positionsalong the cell periphery or surface of the BHK-21 cells (FIGS. 2A and2B). The antigen deposition appeared to be distributed randomly on thecell surface. After 10 minutes, the amount of extracellular fluorescencewas diminished by approximately 40% resulting in fewer areas of antigendeposition. A further reduction in surface-localized fluorescence wasobserved at 30 minutes.

Intracellular Invaplex-Specific Staining Pattern

In general, intracellular fluorescence increased with time of incubationand was specific to those cells that also exhibited extracellularfluorescent particles. After a 30-minute incubation, the greenfluorescence localized to areas surrounding the cell nucleus in a morediffuse manner (FIG. 2C) when compared to the extracellular fluorescencewhich was isolated in bright fluorescent patches.

TABLE 2 Intracellular and Extracellular Antigen Localization of Sflexneri 2a Invaplex Treated BRK cells 5 min. 10 min. 30 min. SampleObservations Observations Observations Media Only No observablefluorescence No observable fluorescence No observable fluorescence(intra/extracellular) (intra/extracellular) (intra/extracellular)ProA-FITC + Rabbit Serum No observable fluorescence No observablefluorescence No observable fluorescence (Invaplex free) Invaplex 24(66.5 ug/well) Extracellular: Extracellular: Extracellular: Invaplex 50(27.7 ug/well) >60% of cells had distinct, 40-60% of cells had distinct30-40% of cells had distinct small fluorescence patches fluorescentparticles lining fluorescent particles lining at cell peripherydecorating (peppering) cell membranes. The (peppering) cell membranes.The cellular membranes. The average number of particles average numberof particles average number of patches per cell was 1 to 2. per cell was1 to 2. per cell was 2 to 4. Invaplex 24 (66.5 ug/well) Intracellular:Intracellular: Intracellular: Invaplex 50 (27.7 ug/well) <10% of cellshave Low-level >30% of cells have low-level >50% of cells have low-levelintracellular fluorescence. intracellular fluorescence. intracellularfluorescence. Only cells with particles on Only cells with particles onOnly cells with particles on their surfaces exhibited their surfacesexhibited their surfaces exhibited intracellular fluorescence. interiorfluorescence. interior fluorescence.

Adherence of S. sonnei Invaplex 24 and 50 with Mammalian Cell Membranes

Similarly to Invaplex 24 and 50 from S. flexneri 2a, Invaplex fromShigella sonnei adheres to BHK-21 cells and is internalized after 5 to30 minutes. Invaplex can be found throughout the cytoplasm of the cell,with a strong presence near the nuclear membrane. (FIG. 3(A-F), FIG.4(A, B), and FIG. 5)

FIGS. 3A, 3C, and 3E are BHK-21 cells viewed under a bright fieldmicroscope (Nikon Optiphot 2) at 20× magnification. The same field isthen shown in FIGS. 3B, 3D, and 3F (Nikon Optiphot 2 and EX470-490excitation filter at 20× magnification. Cells in FIGS. 3A and 3B wereincubated with MEM supplemental with 1% L-glut and 7% FCS alone, cellsin FIGS. 3C and 3D were treated with S. sonnei Invaplex 24, and cells inFIGS. 3E and 3F with S. sonnei Invaplex 50. Bound Invaplex was detectedwith anti-mouse S. sonnei followed by a rhodamine labeled ant-mouse IgG.(Data taken from experiment dated 26 Sep. 2002 in lab notebook titled“In vitro Invaplex Experiments—Volume I, 22 Mar. 2001 to Present”)

BHK-21 fibroblast cells alone (FIG. 4A) or incubated with S. sonneiInvaplex 24 (FIG. 4B) for 5 minutes, fixed with acetone, washed andincubated with mouse anti-Shigella sonnei antibodies which were detectedusing anti-mouse-TRITC. Actin is stained with phalloidin-FITC and thenucleus with propidium iodide.

BHK-21 fibroblasts were treated with S. sonei Invaplex 50 for 10minutes, fixed with methanol, and incubated with anti-Shigella sonneiantibodies raised in mice. After washing thoroughly, wells wereincubated with anti-mouse antibodies conjugated to FTTC. Cells werecounter stained with Evan's Blue. S. sonnei Invaplex 50 can be seen onthe cell's periphery and in the cytoplasm. See FIG. 5.

Intracellular Co-Localization Experiments

To study the interactions between mammalian host cell organelles andShigella Invaplex, BHK-21 fibroblast or HeLa epithelial cells wereincubated with Invaplex for various times, were fixed for 10 minuteswith 10% formalin and permeabilized with 0.1% saponin.² Cells were thenprobed with antibodies specific for Invaplex and intracellularorganelles or vesicles, to include early endosomes (rabbit anti-EarlyEndosomal Antigen (EEA-1); Affinity BioReagents) (28), late endosomes(rabbit anti-Rab 9; Affinity BioReagents) (29, 30), and the Golgiapparatus (mouse anti-58k protein, Sigma) (31, 32). After extensivewashes with 0.1% saponin in PBS, the cells were incubated with secondaryantibodies (Goat anti-Mouse or anti-Rabbit-IgG labeled with eitherOregon Green or Texas Red; Molecular Probes). The washed cells were thenexamined at 60× magnification with a Nikon Optiphot-2 microscopeequipped with green, and red bandpass emission/excitation filter sets. ²BHK-21 fibroblasts were incubated overnight at 37° C. in 8 well glasschamber slides, washed twice with PBS, and incubated with S. flexneriInvaplex-24 for 15 minutes at 37° C. Cells were then washed three timeswith PBS and fixed for 10 minutes with 10% formalin. Fixed cells wereprobed with polyclonal mouse antibodies specific for Shigella Invaplexantigens and polyclonal rabbit antibodies specific for early endosomes(EEA-1) and late endosomes (Rab9). Bound antibodies were subsequentlydetected with GAM-IgG-TRITC (KPL) or GAR-IgG-FITC (KPL). The cells wereexamined at 60× magnification with Nikon Optiphot-2 microscope equippedwith green, and red bandpass emission/excitation filter sets. Imageswere captured with a Pixera 600CL cooled CCD camera and processed forpublication in Photoschop 7.

Results from these studies indicate that Invaplex co-localized withvarious host cell organelles depending on the duration of incubation.Invaplex is first found in early endosomes and appears as punctuatedareas of activity in the cytoplasm. Later the activity “migrates”towards the nucleus and co-localizes with late endosomes (see table 3and FIG. 6A-F). Next the Invaplex activity co-localizes with the Golgiapparatus in a perinuclear staining pattern. Finally, the pattern ofInvaplex staining appears diffusely in the cytoplasm indicating releasefrom either the late endosomes or the Golgi.

TABLE 3 Time points of Invaplex-Intracellular Organelle MarkerCo-localization Early Late Golgi Invaplex Time Endosomal Endosomalapparatus free in point Markers Markers Markers Cytoplasm  1 min +  5min + + 15 min + + 30 min + + 60 min + +

The data in the above table was compiled from multiple experimentsinvestigating the intracellular localization of Invaplex after variousincubation times with mammalian host cells. Each series of experimentsfocused on individual intracellular organelle markers co-localizing withInvaplex at specific incubation times.

Possible Implications of Intracellular Invaplex

There are several possible outcomes of Invaplex-mediated uptake intohost cells, dependent on the cell type. Invaplex-mediated uptake intonon-polarized epithelial cells such as those used in the experimentspresented in Table 3 could result in the presentation of Invaplexantigens in the context of MHC class I molecules. This would beimportant to the adjuvanticity of Invaplex.

Invaplex-mediated uptake into polarized epithelial cells could result inpresentation of Invaplex-antigens to the underlying lymphoid cells viaMHC class I pathway. Alternatively, transport of Invaplex from theapical to the basolateral surface could be accomplished through thesorting mechanism of apically and basolaterally-derived endosomes.

Invaplex-mediated uptake into antigen presenting cells of the immunesystem could result in the presentation of Invaplex, and co-deliveredantigens, via the MHC class I or MHC class II pathway, depending onwhether Invaplex and the antigens escape from the endosomal vacuoles.

Use of Invaplex to Mediate Transfection of Mammalian Cells with PlasmidDNA Encoding the Green Fluorescent Protein

Both S. flexneri 2a Invaplex 24 and Invaplex 50 were capable ofstimulating uptake of plasmid DNA. The level of DNA uptake andsubsequent expression was greater in cells incubated with a higherconcentration (20 μg) of Invaplex as compared to 10 μg of Invaplex. Thelevel of plasmid DNA uptake was determined by the relative amounts offluorescence in the cells. See FIG. 7(A, B).³ ³ BHK-21 cells wereincubated with plasmid DNA (Gene Therapy Systems, San Diego, Calif.)with the green fluorescent protein (GFP) gene linked to a CMV promoter(FIG. 7A) or GFP-plasmid DNA and Invaplex 24 (FIG. 7B) or plasmid DNAand Invaplex 50 (data not shown) overnight at 37° C. GFP expression wasdetermined by the amount of green fluorescence localized in thecytoplasm of the transfected cells. The image was recorded with a NikonOptiphot 2 and EX470-490 excitation filter at 20× magnification.

Cells incubated with GenePorter/plasmid DNA mixtures showed the highestlevel of GFP expression. However, BHK cells treated with the GenePorterTransfection reagent were rounded and not fully extended indicating alevel of cytotoxicity whereas the Invaplex treated cells wheremorphologically indistinguishable from the non-treated cells.

TABLE 4 Relative Amount of Green Fluorescence in BHK cells Transfectedwith plasmid DNA encoding GFP No Transfection Invaplex 24 Invaplex 24Invaplex 50 Invaplex 50 Reagent (10ug) (20 ug) (10 ug) (20 ug)GenePorter GFP − ++ +++ ++ +++ +++++ Plasmid No GFP − − − − − − Plasmid

Invaplex-Mediated Transfection of Mammalian Cells with Plasmid DNAEncoding β-gal.

BHK-21 cells were efficiently transfected with plasmid DNA, encoding theβ-galactosidase gene under the control of a CMV promoter, when incubatedwith a mixture of plasmid DNA and Invaplex 24 or Invaplex 50.⁴ Similarto the previous section, uptake of plasmid DNA was greater with a higherconcentration of Invaplex (20 μg) as compared to transfection with 10 μgof Invaplex. The level of transfection was determined by counting thenumber of cells expressing β-galactosidase. See FIG. 8(A-C). ⁴ BHK-21cells were incubated with β-gal protein (FIG. 8A) or β-gal protein andInvaplex 24 (FIG. 8B) or β-gal protein and Invaplex 50 (FIG. 8C)overnight at 37° C. B-gal protein activity was determinedcolormetrically through the addition of X-gal substrate (Gene TherapySystems, San Diego, Calif.). Cells expressing β-gal activity have a bluecytoplasm.

There was significantly less cytopathic effect when Invaplex was used asa transfection reagent as compared with GenePorter. In the presence ofInvaplex, cells remained adherent and morphologically indistinguishablefrom non cells.

TABLE 5 Relative Amount of β-galactosidase activity (blue color) in BHKcells Transfected with DNA Plasmid encoding β-galactosidase NoTransfection Invaplex 24 Invaplex 24 Invaplex 50 Invaplex 50 Reagent (10ug) (20 ug) (10 ug (20 ug) GenePorter β-gal − + +++ + +++ ++++ plasmidNo β-gal − − − − − − plasmid

Invaplex-Mediated Transport of Purified Green Fluorescent Protein (GFP)Across Mammalian Cell Membranes

The highest percentage of GFP positive cells was found to be in wellswith the lowest amount of Invaplex (Table 9). Fluorescent activity waslow in all of the positive cells indicating transport of active GFP. Thelevel of GFP activity was higher in cells transfected with GFP plasmidas compared to cells treated with Invaplex-GFP protein mixtures which islikely due to continued expression of the gfp gene once inside the cell,resulting in a higher amount of GFP protein in the cell. Also ofinterest was the apparent loss of cells in wells incubated with higherconcentrations of Invaplex in combination with GFP. This may be due toan unknown toxicity of the GFP on mammalian cells or due to an effect ofInvaplex on the adherence mechanism of BHK cells. See FIG. 9(A-F).⁵

TABLE 6 Percentage of GFP-Positive BHK cells Treated with GFP proteinand Invaplex. Media GFP Invaplex 24 Invaplex 50 Only Only 66 μg 33 μg 15μg 28 μg 14 μg 7 μg Amount +++++ +++++ ++ +++ ++++ ++ +++ ++++ ofAdherent Cells Percent 0% 2% 17% 34% 47% 12% 43% 57% GFP+ Cells

Invaplex-Mediated Transport of β-gal Protein Across Mammalian CellMembrane

There was an inverted trend in the amount of adherent cells in a welland the amount of Invaplex-B-gal mixture incubated with those cells. Ingeneral, higher amounts of the Invaplex B-gal mixture resulted in loweramounts of adherent cells at 24 hours (Table 8). This could be theresult of an excess of β-gal being imported into the cell resulting intoxicity or an effect of Invaplex on the adherence mechanism of BHKcells. See FIG. 10(A).

FIG. 8A shows plasmid DNA encoding the β-gal protein incubated withBHK-21 cells (negative control). FIG. 8B shows plasmid DNA encoding theβ-gal protein and S. flexneri 2a Invaplex 24 incubated with BHK-21 cells(Most of the cells have a light blue color in the cytoplasm indicatingbeta-galactosidase expression). FIG. 8C shows plasmid DNA encoding theβ-gal protein and S. flexneri 2a Invaplex-50 incubated with BHK-21 cells(The cells with a light blue color in the cytoplasm indicatebeta-galactosidase expression).

TABLE 7 Percentage of β-gal Positive Invaplex-Treated BHK cells MediaGFP Invaplex 24 Invaplex 50 Only Only 66 μg 33 μg 15 μg 28 μg 14 μg 7 μgAmount +++++ +++++ + ++ ++++ + ++ ++++ of Adherent Cells Percent 0% 0.1%10% 28% 34% 8% 22% 46% GFP+ Cells

Inhibition of Invaplex Adherence to BHK Cells with Shigella Antibodies

Invaplex adherence to mammalian cell membranes was inhibited bypolyclonal serum raised against whole Shigella and by polyclonal serumreactive with IpaC. Polyclonal antibodies reactive with only IpaB orserum from naive animals did not significantly inhibit the adherence ofInvaplex to mammalian cell membranes. These data suggest that theability of Invaplex to bind to host cell membranes may requireinteractions between IpaC and host cell surface structures.

TABLE 8 Inhibition of S. flexneri 2a IVP-24 internalization into BHK-21cells with polyclonal mouse sera or monoclonal antibodies specific forShigella flexneri 2a antigens S. flexneri 2a IVP-24 Blocking AntibodyPercent Cells % Inhibition (μg/ml) Specificity Invaplex-Positive^(a)(Enhancement)^(b) 0 N/A   0% 0.0% 0 S. flexneri 2a IVP-24   0% 0.0% 100N/A 56.6% 0.0% 100 Non (Pre-Bleed) 53.8% 4.6% 100 S. flexneri 2a IVP-24 5.7% 90.6% 100 S. flexneri 2a IVP-24 10.7% 83.1% 100 S. flexneri 2aIVP-50 11.4% 82.6% 100 S. flexneri 2a IVP-50 17.7% 72.8% 100 IpaB 33.3%40.6% 100 IpaC  4.8% 91.5% 100 S. flexneri 2a LPS 58.1% (2.4%)The ability of polyclonal mouse sera, with specificity for S. flexneri2a IVP-24 and IVP-50 antigens, and monoclonal antibodies specific forIpaB, IpaC, or LPS to inhibit the internalization of Shigella Invaplexwas investigated by incubating serum from immunized mice or monoclonalantibodies with S. flexneri 2a IVP24. The antibody-Invaplex mixtureswere then incubated at 37° C. for 30 minutes in duplicate with separateBHK-21 fibroblast monolayers. Controls for the assay included monolayerstreated with mouse antibody in the absence of Invaplex (negativecontrol) and monolayers treated with Invaplex in the absence of antibody(positive control). After incubation, the monolayers were washed withPBS to remove noncell-associated Invaplex and fixed with formalin. Theinternalization of Shigella Invaplex into BHK-21 cells was detected byprobing the formalin-fixed cells with polyclonal rabbit sera specificfor Shigella Invaplex antigens (Rabbit 7) for 2 hours at RT. Themonolayers were washed and bound anti-Invaplex mouse antibodies weredetected with fluorescently-labeled anti-rabbit IgG. Epifluorescentmicroscopy was used to score a minimum of 100 cells per monolayer asbeing either Invaplex-positive or Invaplex-negative based on thepresence or absence of cell-associated Invaplex-specific fluorescence,respectively. The mean number of cells counted and the mean number offluorescent cells in duplicate monolayer treated in the same manner wasreported.

Invaplex-Mediated Transport of Orientia tsutsugamushi 56k Protein andPlasmid DNA Encoding Sta56k Protein Across Mammalian Cell Membrane InVitro

BHK-21 cells were incubated with r56k protein (FIGS. 11A and 11B) orplasmid DNA encoding the r56k protein and Invaplex 24 (FIGS. 11C and11D) or r56k protein and Invaplex 24 (FIGS. 11E and 11F). Cellspossessing r56k protein have a red fluorescence and were photographedusing an Optiphot-2 microscope equipped with a 540 nm excitation filterat 100× magnification.

Results indicate Invaplex is able to facilitate the delivery of plasmidDNA encoding the 56k protein in transcriptionally active form to BHK-21fibroblasts (FIGS. 11 c and 11 d). Furthermore, purified 56k protein canbe translocated to the cytoplasm when incubated with BHK-21 cells in thepresence of Invaplex (FIGS. 11 e and 11 f), but 56k protein does notcross cellular membranes in the absence of Invaplex (FIGS. 11 a and 11b) nor does plasmid DNA encoding the 56k protein (data not shown).

A series of experiments were designed and executed to evaluate theability of Shigella Invaplex to function as a mucosal adjuvant forDNA-based vaccines. Plasmid DNA encoding the sta56 protein was used.

The Sta56 protein from Orientia tsutsugamushi was used as a modelantigen in the two as a model antigen in the two gene immunizationanimal experiments described [sic] below.

Functionality of Shigella Invaplex as a Mucosal Adjuvant for DNA-BasedVaccines Outlined Below. Plasmid DNA Encoding the sta56 Gene was Used asthe DNA Vaccine Construct. Invaplex-Sta56 DNA Adjuvanticity Study.

Humoral Immunity

After three immunizations with plasmid DNA (encoding the Sta56 protein)formulated with and without Invaplex, no detectable anti-Sta56K serumIgG responses were present at one or two weeks post the thirdimmunization (FIG. 12). Immunization with DNA encoding Sta56 proteinformulated with Invaplex and subsequently boosted once with purifiedr56K protein plus Invaplex elicited anti-Sta56 serum IgG responses withendpoints ranging from 1:360 to 1:1440. There was no anti-Sta56 serumIgG responses detectable after one immunization with the Sta56 protein(data not shown). Plasmid DNA immunization without Invaplex formulationdid not substantially prime the humoral immune response as evidencedwith no detectable anti-Sta56 antibody after protein boost. Combined,these results indicate that mucosal immunization with DNA formulatedwith Invaplex primed the humoral immune response to Sta56.

Cell-Mediated Immunity

Significant antigen-specific proliferation was only detected in animalsvaccinated with Invaplex formulated with plasmid DNA encoding the Sta56protein. Plasmid DNA alone or saline control animals did not possessdetectable proliferative responses when stimulated with Sta56 protein(FIG. 13).

Finally the appearance of Invaplex free in the cytoplasm at 60 minutesindicates that it has the ability to escape from the late endosomes orGolgi apparatus. Proteins or nucleic acids co-delivered with Invaplexwould also be released into the host cell cytoplasm.

Functionality of Shigella Invaplex as a Mucosal Adjuvant for DNA-BasedVaccines: Invaplex-Sta56 DNA Adjuvanticity Study II

The ability of Shigella Invaplex to enhance the immunogenicity of aplasmid DNA-based vaccine was evaluated in mice. Groups of femaleBalb/cByJ mice (10 mice/grp) were intranasally immunized on day 0, 14,and 28 with plasmid DNA containing the sta56 gene (25) from the Karpstrain of Orienta tsutsugamushi linked to a cytomegalovirus (CMV)promoter (pVR1012_sta56). Mice were immunized with pVR1012_sta56 alone(25 or 100 μg) or pVR1012_sta56 combined with S. flexneri 2a Invaplex-50(15 μg). Controls for the study included groups of mice intranasallyimmunized with either saline, S. flexneri 2a IVPlnvaplex-50 (15 μg) orthe empty expression vector (pVR1012) (100 μg) combined with S. flexneri2a Invaplex-50 (15 μg). Animals (5 mice/group) were boosted on day 56with an intranasal immunization of purified recombinant Sta56 protein(15 μg) combined with S. flexneri 2a Invaplex-50 (5 μg). Animals werebled from the tail before vaccination on day 0 and day 28, and on days35, 42, 56, 63, and 70. Cervical lymph nodes (CLN) and spleen cells werecollected on day 70. Antigen-specific antibody responses were assessedin serum samples by an enzyme linked immunosorbant assay (ELISA) aspreviously described (27). Coating concentrations of the variousantigens plated at 50 μl/well were: S. flexneri 2a Invaplex-50 (1 μg/ml)and the Sta56 protein (3 μg/ml). Splenocytes and cells from cervicallymph nodes were evaluated for antigen-specific proliferation using acolorimetric assay. Antigens used for proliferation included the Sta56protein (20 μg/ml or 5 μg/ml) or S. flexneri 2a Invaplex-50 (5 μg/ml or1 μg/ml).

Cell-Mediated Immune Responses Elicited with Plasmid DNA Encoding theSta56 Protein from O. tsutsugamushi (pVR1012 56K) Delivered Alone or inCombination with S. flexneri 2a Invaplex-50

Spleens were harvested on day 42, two weeks after the third DNAimmunization, from 5 mice of each treatment group. Antigen-specific(Sta56 and S flexneri 2a Invaplex-50) and mitogenic (Con A induced)proliferation was measured using a colorimetric proliferation assay.Whereas splenocytes from all animals from each treatment groupproliferated in response to in vitro stimulation with Con A (FIG. 14,Top Panel), only animals immunized with pVR1012_(—)56K combined with S.flexneri 2a Invaplex-50 proliferated after stimulation with purified,Sta56 protein (FIG. 14, Bottom Panel). The Sta56-specific proliferativeresponse after immunization with pVR1012_(—)56K (25 or 100 μg) combinedwith S. flexneri 2a Invaplex-50 was significantly higher (p<0.002) thanthe Sta56-specific proliferative responses detected after immunizationwith pVR1012_(—)56K (100 μg) alone. The mean stimulation index (SI) ingroups of mice immunized with pVR1012_(—)56K (25 μg) combined with S.flexneri 2a IVP-50 was comparable (p=0.12) to the mean SI from miceimmunized with pVR1012_(—)56K (100 μg) combined with S. flexneri 2aInvaplex-50, indicating that intranasal immunization with higher amountsof pVR1012_(—)56K combined with S. flexneri 2a Invaplex-50 did notresult in higher levels of Sta56-specific proliferation (FIG. 14, MiddlePanel).

Invaplex-Specific Proliferation in Splenocytes After Immunization withPlasmid DNA and Invaplex.

The Invaplex-specific proliferative responses were also measured afterimmunization with plasmid DNA and Invaplex. (FIG. 14, Middle Panel).Splenocytes isolated from mice immunized with S. flexneri 2a Invaplex-50alone, or S. flexneri 2a Invaplex-50 combined with pVR1012 or S.flexneri 2a Invaplex-50 combined with pVR1012_(—)56K proliferated afterin vitro stimulation with S. flexneri 2a Invaplex-50, whereas nodetectable Invaplex-specific proliferation was detected in groupsimmunized with saline or pVR1012_(—)56K alone, further demonstrating theinduction of Invaplex-specific cell-mediated immunity after immunizationwith S. flexneri 2a Invaplex-50.

Enhancement of Sta56-Specific Antibody Responses Elicited After ShigellaInvaplex-Mediated Mucosal Delivery of Plasmid DNA-Based Vaccines In Vivowith an Invaplex-Sta56 Protein Booster Immunization.

Immunization with plasmid DNA encoding vaccine antigens has beenpreviously shown to elicit primarily an antigen-specific T cell-mediatedimmune response and the generation of weak antigen-specific antibodyresponses (24). The enhancement of weak vaccine-specific antibodyresponses after DNA immunization has been previously accomplished byfollowing the DNA vaccine regimen with a protein-based boosterimmunization (DNA prime-protein boost regimen) (24, 26).

Anti-Sta56 Serum Antibody Responses After Immunization withpVRIO12_(—)56K Delivered Alone, or pVRIO12_(—)56K Co-Delivered with S.flexneri 2a IVP-50 Followed by a Sta56 Protein-Invaplex BoosterImmunization.

Intranasal immunization with saline on day 0, 14, and 28 followed byintranasal immunization with Sta56 protein (15 μg) combined with S.flexneri 2a Invaplex-50 (Invaplex-Sta56 protein) on day 56 did notresult in the generation of detectable anti-Sta56 serum IgG responses(FIG. 15-A) indicating that one dose of Invaplex-Sta56 protein was notsufficient to mount a robust Sta56-specific humoral immune response. Incontrast, groups of mice intranasally immunized three times withpVR1012_(—)56K (either 25 or 100 μg) delivered with S. flexneri 2aInvaplex-50 (15 μg) followed by a single Invaplex-Sta56 protein boosterimmunization mounted a modest Sta56-specific serum IgG response detectedone and two weeks after the protein booster immunization. The Sta56specific serum IgG response elicited after intranasal immunization withpVR1012_(—)56K (either 25 or 100 μg) delivered with S. flexneri 2aIVP-50 (15 μg) was significantly higher (p<0.05; unpaired T test) ascompared to the Sta56-specific serum IgG response elicited afterimmunization with pVR1012_(—)56K alone one and two weeks after theInvaplex-Sta56 protein booster immunization. Immunization with Sflexneri 2a Invaplex-50 alone, or combined with pVR1012 (emptyexpression vector control) did not result in a detectable anti-StaS6serum IgG response one or two weeks after the Invaplex-Sta56 proteinboost, indicating that successful priming of the immune system withpVR1012_(—)56K delivered with Invaplex was required for the subsequentSta56-specific antibody response post-protein boost.

Anti-Invaplex Antibody Responses After Immunization with pVR1012_(—)56KDelivered Alone, or pVR1012_(—)56K Co-Delivered with S. flexneri 2aInvaplex-50 Followed by an Invaplex-Sta56 Protein Booster Immunization(Subgroup B).

The Invaplex-specific serum IgG responses were also assessed by ELISA(FIG. 15-B). Groups of mice immunized with saline or pVR1012_(—)56Kalone did not mount a detectable anti-Invaplex serum IgG response at anytime point assayed, including one and two weeks after the singleInvaplex-Sta56 protein boost. Groups of mice immunized with S. flexneri2a delivered alone, or in combination with pVR1012 or pVR1012_(—)56K (25or 100 μg) mounted a robust anti-Invaplex serum IgG response, firstdetected one week after the third immunization (day 35). Theanti-Invaplex serum response increased on day 42 and was subsequentlyboosted on day 63, one week after the Invaplex-Sta56 protein boosterimmunization to levels similar to those levels measured two weeks afterthe third immunization (day 42). The anti-Invaplex serum IgG responses,after the third immunization, were maintained at high levels, withmodest decreases in the anti-Invaplex serum IgG responses over the onemonth period between the third and fourth immunization, indicating thegeneration of a robust anti-Invaplex response.

Collectively, the results of the Invaplex-56K DNA experimentsdemonstrate that three intranasal immunizations with S. flexneri 2aInvaplex-50 combined pVR1012_(—)56K elicits a 56K-specific andInvaplex-specific cell-mediated immune response. Boosting of the immuneresponse with an intranasal immunization consisting of Invaplex combinedwith Sta56 protein (DNA-prime, protein-booster immunization regimen)broadened the Sta56-specific immune response to include a modestanti-Sta56 serum IgG response combined with Sta56-specific cell-mediatedimmunity measured in splenocytes two weeks after the fourthimmunization. Therefore, data from the second Invaplex Sta56 DNAAdjuvanticity study confirm previous results and indicate that S.flexneri 2a Invaplex-50 was capable of enhancing the immunogenicity of aplasmid-based DNA vaccine and acts as a mucosal adjuvant for DNA-basedvaccines.

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To the extent necessary to understand or complete the disclosure of thepresent invention, all publications, patents, and patent applicationsmentioned herein are expressly incorporated by reference therein to thesame extent as though each were individually so incorporated.

1. An in vitro process for transporting a compound of interest into aeukaryotic cell comprising contacting the eukaryotic cell with thecompound and a sufficient amount of isolated Invaplex and for a timesufficient to cause the cell to take up the compound, wherein theInvaplex has the composition of either Invaplex 24 or Invaplex
 50. 2.The in vitro process of claim 1 wherein the compound is an antibiotic,antibody, biopharmaceutical, hormone reporter molecule, enzyme orreceptor.
 3. The in vitro process of claim 1 wherein the compound is acarbohydrate, glycoprotein, lipid, lipopolysaccharide, polysaccharide,protein or peptide.
 4. The in vitro process of claim 3 wherein thecompound is a protein.
 5. The in vitro process of claim 3 wherein thecompound is a peptide.
 6. The in vitro process of claim 1 wherein thecell is a tumor cell.
 7. The in vitro process of claim 2 wherein thecompound is an enzyme.
 8. An in vivo process for transporting a compoundof interest into eukaryotic cells of a subject comprising separatelyadministering the compound and an effective amount of isolated Invaplexto the subject, wherein the Invaplex has the composition of eitherInvaplex 24 or Invaplex 50 and wherein the compound is a carbohydrate,glycoprotein, lipid, lipopolysaccharide, polysaccharide, protein orpeptide and monitoring the presence of the compound within the cells. 9.The in vivo process of claim 8 wherein the compound is a protein. 10.The in vivo process of claim 9 wherein the compound is an enzyme. 11.The in vivo process of claim 8 wherein the compound is a peptide. 12.The in vivo process of claim 8 wherein the administration involvesmucosal administration.